title: Selection and validation of endogenous controls for microRNA expression studies in endometrioid endometrial cancer tissues.
abstract: OBJECTIVE
  microRNAs comprise a family of small, non-coding RNAs, which regulate gene expression at the posttranscriptional level. Multiple studies implicated important roles of microRNAs in various malignancies including endometrioid endometrial carcinoma (EEC). qPCR is widely used in the studies investigating microRNA expression. Relative quantification of microRNA expression requires proper normalization methods and endogenous controls are widely used for this purpose. The aim of this study was experimental identification of stable endogenous controls for normalization of microRNA qPCR expression studies in EEC.
  
  
  METHODS
  Expression of twelve candidate endogenous controls (miR-16, miR-26b, miR-92a, RNU44, RNU48, U75, U54, U6, U49, RNU6B, RNU38B, U18A) was investigated in tissue samples obtained from 45 patients (30 EEC, 15 normal endometrium) using qPCR. Stability of candidate endogenous controls was evaluated using NormFinder, geNorm, BestKeeper and equivalency test. The results were then validated using larger group of samples.
  
  
  RESULTS
  RNU48, U75 and RNU44 were identified as stably and equivalently expressed between malignant and normal tissues. Both NormFinder and geNorm indicated that those three snRNAs were optimal for qPCR data normalization in EEC tissues.
  
  
  CONCLUSIONS
  In conclusion, we suggest that average expression of those snoRNAs could be used as a reliable endogenous control in microRNA qPCR studies in endometrioid endometrial cancer. In addition to identifying suitable endogenous controls in EEC, our study presents an appropriate strategy for validation of candidate reference genes for any microRNA qPCR study.
authors.affiliation: Laboratory of Biostructure, Department of Human Anatomy, Medical University of Lublin, Jaczewskiego 4, 20-090, Lublin, Poland. anna.torres@wp.pl
authors.forename: Anna
authors.initials: A
authors.lastname: Torres
authors.forename: Kamil
authors.initials: K
authors.lastname: Torres
authors.forename: Paulina
authors.initials: P
authors.lastname: Wdowiak
authors.forename: Tomasz
authors.initials: T
authors.lastname: Paszkowski
authors.forename: Ryszard
authors.initials: R
authors.lastname: Maciejewski
mesh_terms: D018269:Carcinoma, Endometrioid; D016889:Endometrial Neoplasms; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar
keywords: Endogenous control; Endometrioid endometrial cancer; MicroRNA; Real-time qPCR; Reference gene